Glycosyl dihydrogenphosphate salts are key intermediates in the biological synthesis of nucleotide sugars that are involved in the assembly of oligosaccharide chains of glycoproteins and glycolipids. Glycosyl dihydrogenphosphate salts in the presence of nucleoside triphosphates are converted to nucleotide sugars by the enzyme nucleotide sugar synthetase. Once formed, these nucleotide sugars function as the donor substrates for glycosyltransferases, which transfer an .alpha. or .beta. glycosyl residue to growing oligosaccharide acceptor substrates. Enzymatic modification of cell surface oligosaccharide structures using two key tools, the nucleotide sugars and glycosyltransferases, has been shown to be very useful for investigating the role of carbohydrates on these macro molecules. Since several glycosyltransferases have been purified and are commercially available, the preparation of the structurally diverse nucleotide sugar substrates for these enzymes is required for the above mentioned biological investigations.
Many nucleotide sugars have been made enzymatically, and some of these, particularly the natural sugar nucleotides, are also available commercially at a very high cost. Yet, the structural diversity in the glycosyl residues available from these sources is limited, as the enzymatic preparation of nucleotide sugars is dependent on the substrate specificity of the nucleotide-sugar synthetase enzymes.
Chemical methods are an attractive alternative for the preparation of structurally diverse nucleotide sugar derivatives. However, such methods would still require the ready availability of glycosyl dihydrogenphosphate salts that can be coupled to the activated nucleotide mono- or diphosphates. Thus, there is a need for processes for the preparation of glycosyl dihydrogenphosphates and its salts.
Several reports have appeared which disclose the preparation of glycosyl phosphate triesters, or their dihydrogenphosphates and salts of specific sugars using various reagents
Prihar, H. S., et al., Biochemistry, Vol. 12, 997 (1973) and Nunez, H. A., et al., Can. J. Chem., Vol. 59, 2086 (1981) disclose preparation of glycosyl phosphate esters using O-phenylene phosphorochloridate.
Inage, M., et al., Chem. Letters, 1281 (1982) and Yamazaki, T., et al., Can. J. Chem. Vol. 59, 2247 (1981) teach preparation of glycosyl phosphate esters using dibenzyl chlorophosphate and butyl lithium.
Dibenzyl phosphorofluoridate synthesis and its use as a phosphorylating agent is disclosed by Watanabe, Y., et al., Tetrahedron Letters, Vol. 29, 5763 (1988).
The preparation of D-glucopyranosyl phosphates from D-glucopyranosyl trichloroacetimidates is reported by Schmidt, R. R., et al., Tetrahedron Letters, Vol. 23 405 (1982).
Granata, A , et al., Carbohydr. Res., Vol. 94, 165 (1981) disclose the use of diphenyl chlorophosphate and thallium ethoxide or n-butyl lithium in the synthesis of phosphate and related ester derivatives of carbohydrates.
Hashimoto, S., et al., J. Chem. Soc. Chem. Commun., 685 (1989) report a rapid synthesis of 1,2-trans-beta-linked glycosides via benzyl- or benzoyl-protected glycopyranosyl phosphate triesters.
The use of hexopyranosyl acetates and phosphoric acid, as well as the use of glycosyl orthoesters and dibenzyl hydrogenphosphate have also been reported in the preparation of glycosyl phosphate esters.
The object of the present invention is to provide a process for the preparation of glycosyl phosphates in their triester form, preferably anomerically enriched starting with the readily available hexopyranose compounds and the commercially available diphenyl chlorophosphate and 4-N,N-dimethylaminopyridine (DMAP). The glycosyl phosphate triesters prepared via this process can be converted to the natural glycosyl monohydrogenphosphate salts suitable for reaction with activated nucleoside mono- or diphosphates.